Background. Pulmonary segmentectomy is being increasingly used to resect small lung nodules; however, identifying the intersegmental plane is difficult. We describe a new methylene blue staining technique that we developed to identify the intersegmental planes in anatomic segmentectomy using video-assisted thoracic surgery (VATS) or thoracotomy and to evaluate its feasibility and safety. Methods. Between October 2013 and December 2013, 14 consecutive patients with lung disease underwent anatomic segmentectomy at our institution (10 VATS, 4 conventional thoracotomy). Methylene blue 0.1% (20 mL) was slowly injected into the bronchus of the target pulmonary segments using an intravenous needle after division of the artery, vein, and bronchus of the target segments, and the boundaries were detected, followed by anatomic segmentectomy. Results. The staining took only 3 min. The target pulmonary segments stained blue, allowing for the clear identification of the intersegmental plane on both the surface and in the lung parenchyma, and all operations were successfully completed. Staining did not affect pathologic examination of the resected specimens. The fluid that drained from the chest tube and the patients' sputum, urine, and feces were not blue. There were no perioperative deaths or major complications. Conclusions. To our knowledge, this study is the first to report a safe and feasible methylene blue staining method for identifying the lung segment borders that does not require any special equipment. More importantly, this method can clearly detect the intersegmental planes on the pleural surface and within the lung parenchyma, enabling thoracic surgeons to accurately perform anatomic segmentectomy. (C) 2015 by The Society of Thoracic Surgeons